The significance of cytogenetics for the study of karyotype evolution and taxonomy of water bugs (Heteroptera, Belostomatidae) native to Argentina

CompCytogen 7(2): | 11-129 (2013) COMPARATIVE A reerrerewet open-access over

doi: 10.3897/CompCytogen.v7i2.4462 Kan Cyto genetics

www.pensoft.net/journals/compcytogen International journal of Plant & Animal Cytogenetics,

Karyosystematics, and Molecular Systematics

The significance of cytogenetics for the study of
karyotype evolution and taxonomy of water bugs
(Heteroptera, Belostomatidae) native to Argentina

Chirino Ménica Gabriela'?", Alba Graciela Papeschi'*, Maria José Bressa'’

I Znstituto de Ecologia, Genética y Evolucién de Buenos Aires, Departamento de Ecologia, Genética y Evolu-
cién, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Ciudad Universitaria, Pabellén
I, C1428EHA, Ciudad Auténoma de Buenos Aires, Argentina 2 Laboratorio de Entomologia Aplicada y
Forense, Departamento de Ciencia y Tecnologia, Universidad Nacional de Quilmes, Roque Sdenz Pena 352,
B1876BXD, Bernal, Buenos Aires, Argentina

Corresponding author: Chirino Ménica Gabriela (mchirino@ege.fcen.uba.ar)

Academic editor: S. Grozeva | Received 10 December 2012 | Accepted 19 March 2013 | Published 29 April 2013

Citation: Chirino MG, Papeschi AG, Bressa MJ (2013) ‘The significance of cytogenetics for the study of karyotype
evolution and taxonomy of water bugs (Heteroptera, Belostomatidae) native to Argentina. Comparative Cytogenetics

7(2): 111-129. doi: 10.3897/CompCytogen.v7i2.4462

Abstract

Male meiosis behaviour and heterochromatin characterization of three big water bug species were studied.
Belostoma dentatum (Mayr, 1863), B. elongatum Montandon, 1908 and B. gestroi Montandon, 1903 pos-
sess 2n = 26 + X,X,Y (male). In these species, male meiosis is similar to that previously observed in Belos-
toma Latreille, 1807. In general, autosomal bivalents show a single chiasma terminally located and divide
reductionally at anaphase I. On the other hand, sex chromosomes are achiasmatic, behave as univalents
and segregate their chromatids equationally at anaphase I. The analysis of heterochromatin distribution
and composition revealed a C-positive block at the terminal region of all autosomes in B. dentatum, a C-
positive block at the terminal region and C-positive interstitial dots on all autosomes in B. elongatum, and
a little C-positive band at the terminal region of autosomes in B. gestroi. A C-positive band on one bivalent
was DAPI negative/CMA, positive in the three species. The CMA,-bright band, enriched in GC base
pairs, was coincident with a NOR detected by FISH. ‘The results obtained support the hypothesis that all
species of Belostoma with multiple sex chromosome systems preserve NORs in autosomal bivalents. The
karyotype analyses allow the cytogenetic characterization and identification of these species belonging to
a difficult taxonomic group. Besides, the cytogenetic characterization will be useful in discussions about

evolutionary trends of the genome organization and karyotype evolution in this genus.

Copyright Chirino Monica Gabriela et al. This is an open access article distributed under the terms of the Creative Commons Attribution License
3.0 (CC-BY), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

112 Chirino Ménica Gabriela et al. / Comparative Cytogenetics 7(2): 11 1-129 (2013)

Keywords
Heteroptera, holokinetic chromosomes, karyotype evolution mechanisms, multiple sex chromosomes,

rDNA-FISH

Introduction

Belostomatidae include some of the largest heteropteran species, which are general
predators that play an important role as biological agents in aquatic environments
(Menke 1979, Smith 1997, Saha et al. 2010). This family has a cosmopolitan distribu-
tion in tropical and subtropical areas in the world (Schnack 1976, Polhemus and Pol-
hemus 2008). In South America, the genus Be/ostoma Latreille, 1807 is the most diverse
and includes 61 species mainly distributed from Colombia and Brazil to Argentina
and Chile (Heckman 2011). Nevertheless, cytogenetic reports in Belostoma from South
America comprise the male chromosome complement of 15 species and male meiosis
analysis of 13 species (Table 1). Ten of these species show a modal diploid chromosome
number 2n = 29 = 26 + X,X,Y (male) and five species possess reduced chromosome
numbers and a simple sex chromosome system XY/XX (male/female) (Table 1).

All species of Belostoma analyzed possess holokinetic chromosomes, i.e. chromo-
somes without a primary constriction and therefore without a localized centromere.
Autosomal bivalents are synaptic and chiasmatic, whereas sex chromosomes are asyn-
aptic and achiasmatic, and behave as univalents in first male meiotic division. Howev-
er, at metaphase II sex chromosomes associate end-to-end through the so called touch-
and-go pairing, forming a pseudo-bivalent or pseudo-multivalent. In the first meiotic
division, autosomal bivalents segregate reductionally while sex chromosomes divide
equationally (Ueshima 1979, Papeschi and Bidau 1985, Suja et al. 2000, Papeschi and
Bressa 2006, Bardella et al. 2012). During meiosis, the kinetic activity is restricted to
the chromosome ends and the chromosomes can be regarded as telokinetic (Motzko
and Ruthmann 1984).

Most hypotheses on karyotype evolution in Heteroptera include both autoso-
mal and sex chromosome fusions and fragmentations (Ueshima 1979, Manna 1984,
Thomas 1987, Papeschi 1994, 1996, Pérez et al. 2004). The cytogenetic data available
for Belostoma allow to hypothesize that current karyotypes with a multiple sex chro-
mosome system (X_Y/X_X_, male/female) are derived through fragmentation of the
ancestral X from an XY sex chromosome system. On the other hand, reduced chromo-
some complements with simple sex chromosome system (XY/XX, male/female) have
probably originated through several chromosomal fusions (Papeschi 1996, Papeschi
and Bressa 2006, Bardella et al. 2012).

The aim of this study was to perform a detailed comparison of male meiosis behav-
iour and examine the structure of the holokinetic chromosomes by means of C- and
fluorescent bandings, and fluorescent im situ hybridization (FISH) with 18S rDNA
probes in Belostoma dentatum (Mayr, 1863), B. elongatum Montandon, 1908 and B.
gestroi Montandon, 1903. The female complement and the male meiosis of B. elonga-

The significance of cytogenetics for the study of karyotype evolution and taxonomy... 113

Table |. Diploid chromosome number, chromosome bandings and nucleolar organizer region (NOR)

detected by FISH in South American Belostoma species. *A: autosomal bivalent, **X, Y: sex chromosomes

DAPI/ rDNA
Species Chae Hands te FISH References

Re “ Papeschi and Bressa 2004

B. bifoveolatum Spinola,
1852

B. candidulum
Montandon, 1903 yes -- Bardella et al. 2012

B. Sas ‘a Carlo, 26 +X, XY ia no ah Papeschi and Bidau 1985
; Papeschi and Bidau 1985,
B. dentatum (Mayr, 1863) | 26 + X,X,Y yes A Bipeschi 199d, this seid
26: XN no 23 Papeschi 1992
B.di four, 1
Gaia OEE LEGS) 26 Px KRY yes “4 Bardella et al. 2012

B. discretum Montandon, Papeschi 1992, Chirino and
1903 a AG ues c Bressa 2011

, Papeschi 1988, 1991, Papeschi
26 + X XY A
B. elegans (Mayr, 1871) a ue and Bidau 1985,
a | Papeschi and Bressa 2006

B. elongatum Montandon, F Papeschi and Bressa 2006, this
1908 26 +X XY yes A aud

B. gestroi Montandon, 1903| 26 +X X,Y yes A’ __|Papeschi and Bressa 2006, this study

B. martini (Montandon,
1899) 26+XX,Y no ea Papeschi 1991

Papeschi 1988, Papeschi and

Papeschi 1991, Chirino and
Bressa 2011

B. micantulum (Stal, 1860) yes b e's etn 00G
1 hi 1996, Chiri d

B. orbiculatum Estévez & yes -- ae FA e rae iy

ressa 2011

Polhemus, 2001 :
Papeschi 1996
Papeschi 1988, 1995, Papeschi
B. oxyurum (Dufour, 1863) yes De Wi Batten 2006
no --

B. plebejum (Stal, 1858) Papeschi 1994

Pp 14+XXY ||

tum and B. gestroi are described for the first time. These results allowed us to distin-
guish morphologically similar species and, also, led us to propose a scenario of karyo-
type evolution in the genus Belostoma.

Material and Methods

Insects

For meiotic analysis, adults and nymphs of B. elongatum (9 males and 8 females) and
B. gestroi (4 males and 12 females) were collected from 1988 to 1990 in several fields

114 Chirino Ménica Gabriela et al. / Comparative Cytogenetics 7(2): 11 1-129 (2013)

Table 2. Species, provenience, geographical coordinates, and number of adults’ collected and examined

of Belostoma for chromosomal analyses discriminated by gender.

Species Chromosomal analyses Localities from Argentina | Coordinates |N° of individuals
: 33°40'33"S,
C- and DAPI-CMA, bandings |San Pedro, Buenos Aires 59°39'47"W 3 males
B. dentatum

B. elongatum| Male meiotic behaviour

FISH technique

Chromosome complement

Corrientes, Corrientes

Arroyo Cuay Grande,
Corrientes

Lagos de Stieler, Misiones

Valle Hermoso, Misiones

27°28 16'S,
58°50'22"W
28°28'16'S,
58°50'22"W
26°34'2"S,
54°45'57"W
26°23 10°S,
54°27'58"W

1 female

1 female

1 male

8 males, 7 females

C- and DAPI-CMA, bandings
FISH technique

Chromosome complement

Corrientes, Corrientes

Rio San Pedro, Buenos

Aires

27728 16,5;
58°50'22"W
33°40'33"S,
59°39'47"W

3, males

1 male

31°36'4"S,

60°34'12"W
B°26.007S;
60°22'00"W
27°28 16-95
58°50'22"W

Rincén Norte, Santa Fe 3 males, 11 females

B. gestroi Male meiotic behaviour

1 female

Santa Rosa, Santa Fe

C- and DAPI-CMA, bandings . :
. 3 Corrientes, Corrientes
FISH technique

2 males, 1 female

from Buenos Aires, Santa Fe, Entre Rios, Corrientes and Misiones provinces, all in
Argentina (Table 2). For chromosome bandings and fluorescent in situ hybridization
(FISH) technique, adults of B. dentatum (3 males and 1 female), B. elongatum (3
males) and B. gestroi (2 males and 1 female) were collected from 2010 to 2011 in Cor-
rientes province (Argentina) (Table 2). Collected adults were identified according to
the keys provided by Schnack (1976) and Heckman (2011).

Chromosome preparations

The captured specimens were brought alive to the laboratory and reared until their
gonads were dissected out. For meiotic analysis, the adults and nymphs were fixed for
15-30 min in freshly prepared fixative (ethanol:glacial acetic acid, 3:1). Afterwards,
gonads were dissected out and kept at 4° C in 70% ethanol. Slides were prepared by
the squash technique in a drop of 2% iron-propionic haematoxylin following con-
ventional procedures (Sdez 1960). For C- and fluorescent bandings, and FISH tech-
nique, gonads were dissected in a physiological saline solution for Ephestia Guenée,
1845 (Glaser 1917: cited by Lockwood 1961), swollen for 15 min in a hypotonic
solution (0.075 M KCl), and fixed for 15-30 min in freshly prepared Carnoy fixative

The significance of cytogenetics for the study of karyotype evolution and taxonomy... 15

(ethanol:chloroform:glacial acetic acid, 6:3:1). Spread chromosome preparations were
made in a drop of 60% acetic acid with the help of tungsten needles and spread on the
slide using a heating plate at 45° C as described in Traut (1976). The preparations were
dehydrated in an ethanol series (70, 80 and 96%, 30 sec each) and stored at -20° C
until use.

Chromosome bandings

Heterochromatin content, distribution and nucleotide composition were analysed by
means of C- and sequential fluorescent DAPI and CMA, bandings. C-banding was
performed according to Papeschi (1988), and the pre-treated slides were stained with
4’6-diamidino-2-phenylindole (DAPI; Fluka BioChemika, Sigma Aldrich Production
GmbH, Buchs, Switzerland) for a better resolution of C-bands (Poggio et al. 2011).
Fluorescent banding with AT-specific DAPI and GC-specific chromomycin A, (CMA,;
Fluka BioChemika) was carried out following Poggio et al. (2011).

Fluorescence in situ hybridization

Unlabelled 18S ribosomal DNA (rDNA) probes were generated by polymerase chain
reaction (PCR) using universal arthropod primers: forward 5’-CCTGAGAAACG-
GCTACCACATC-3’ and reverse 5’-GAGTCTCGTTCGTTATCGGA-3’ (Whiting
2002). Total genomic DNA of Dysdercus albofasciatus Berg, 1878, obtained by standard
phenol-chloroform-isoamylalcohol extraction, was used as a template. PCR was done
following the procedure described in Fukova et al. (2005). The PCR product showed a
single band of about 1,000 bp on a 1% agarose gel. The band was recovered from the
gel and purified by using a QIAquick Gel Extraction Kit (Quiagen GmbH, Hilden,
Germany). The 18S rDNA fragment was re-amplified by PCR and then labeled with
biotin 14-dATP by nick translation using a BioNick Labeling System (Invitrogen, Life
Technologies Inc., San Diego, CA, USA). FISH with a biotinylated 18S rDNA probe
was carried out following the procedure in Sahara et al. (1999) with several modifica-
tions described by Fukova et al. (2005) and Bressa et al. (2009).

C-positive heterochromatin and DNA measurements

Data of C-positive heterochromatin percentage and the haploid DNA content in B.
dentatum, B. elongatum and B. gestroi are part of the results obtained by Papeschi in
her Ph.D. Thesis (1992). The procedures of C-positive heterochromatin percentage
and the haploid DNA content were published by Papeschi in 1991 and 1988, respec-
tively. Hence, these results were mentioned only in this paper to analyze and discuss
the extent of karyotype uniformity in these three species. Briefly, the study of the C-

116 Chirino Ménica Gabriela et al. / Comparative Cytogenetics 7(2): 11 1-129 (2013)

positive heterochromatin measurements was performed on meiotic cells. For each spe-
cies at least 10 C-banded cells at diakinesis and without superimposed bivalents were
photographed. The percentage of C-positive heterochromatin was calculated as the
C-positive area divided by the total chromosome area. The DNA content was carried
out by Feulgen microdensitometry (Papeschi 1988) in individuals fixed similar time

ago (1-3 months) (Papeschi 1991).

Statistical analysis

The total chromosome length measurements (TCL) were performed with Micro Meas-
ure for Windows, version 3.3. The TCL of all bivalents and sex chromosomes were
performed in metaphase I. Differences in TCL among species were compared by us-
ing one-way analysis of variance (ANOVA), with Fisher adjusted a posterior contrast.
Statistical analyses were done using Statview software (SAS Institute Inc., 1992-1998).

Microscopy, photographs and image processing

Preparations were observed in epifluorescence microscopes: Zeiss Laborlux (Carl Zeiss,
Germany) equipped with an analogue camera and Leica DMLB equipped with a Leica
DFC350 FX CCD camera and Leica IM50 software, version 4.0 (Leica Microsystems
Imaging Solutions Ltd., Cambridge, UK). Photomicrographs from meiotic chromo-
some preparations were taken using Kodak colour Supra print film 400 ASA. Black-
and-white images of chromosomes from C- and fluorescent bandings and FISH tech-
nique were recorded separately for each fluorescent dye with the CCD camera. Images
were pseudo-coloured (light blue for DAPI, green for CMA,, and red for Cy3), and

processed with an appropriate software.

Results

Male chromosome complement and meiosis

Male meiotic karyotypes based on metaphase I autosomal bivalents (II) and sex uni-
valents of B. dentatum, B. elongatum and B. gestroi show a male diploid chromosome
number 2n = 13] + X,X,Y (Fig. 1). In the three species, the autosomes decrease gradu-
ally in size, both X chromosomes differ slightly in size and the Y chromosome is the
smallest of the complement. The chromosome complement and male meiotic behav-
iour of B. dentatum have already been described (Papeschi and Bidau 1985). ‘The three
species of Belostoma show statistical differences in total chromosome length (TCL) (F ;.
93 = 8.484; P = 0.0004), which is higher in B. dentatum (39.43 + 3.72 um), intermedi-
ate in B. elongatum (37.03 + 2.96 um) and lower in B. gestroi (33.31 + 3.64 um).

The significance of cytogenetics for the study of karyotype evolution and taxonomy... 117

Autosomal bivalents XX Y

ce
ae
as
ae
&
eae
es
Be
&
we
)
te
ae
wo
ae
-

f A q $ Fd ry t 3 % — f —_— — - _ x,
— #

C

Figure |. Male meiotic karyotypes of B. dentatum (a), B. elongatum (b) and B. gestroi (c), 2n = 13] +
X,X.Y, stained with 2% iron-propionic haematoxylin.

Analysis of spermatogonial prometaphase of B. elongatum and B. gestroi revealed
a diploid number of 29 chromosomes; both karyotypes were as described by Pape-
schi (1992) (Fig. 2a). Male meiotic behaviour in B. elongatum and B. gestroi was
similar and followed the same pattern as previously described for other Belostoma
species. Thus, we showed a single and combined Figure 2 with meiotic stages from
both species. At synizesis, the first meiotic identifiable stage of meiosis, chromatin
condenses eccentrically in the nucleus (Fig. 2b). At pachytene, an autosomal bi-
valent is associated with the nucleolus and the 13 autosomal bivalents are joined
through their positive heteropycnotic terminal regions. The condensed sex chro-
mosomes, close to each other, may be distinguished (Fig. 2c). In this cell, the two
X chromosomes have a secondary constriction, but these constrictions are observed
in only one specimen of B. elongatum. During the diffuse stage, all bivalents decon-
dense completely, except for some chromocentres (Fig. 2d). In B. gestroi, at early
diakinesis, both X chromosomes are negative heteropycnotic, and the Y chromo-
some is positive heteropycnotic (Fig. 2e, f). At late diakinesis, the three sex univa-
lents and the 13 autosomal bivalents becomes isopycnotic in both species (Fig. 2g,
h). Each bivalent has a single chiasma in either subterminal or terminal positions
(Fig. 2e—h). At metaphase I, autosomal bivalents arrange in a ring, but the Xs and
Y univalents do not show a defined position (Fig. 2i). During anaphase I, the bi-

118 Chirino Ménica Gabriela et al. / Comparative Cytogenetics 7(2): 111-129 (2013)

Figure 2. Male meiosis in B. elongatum (b, ¢, g, j, k) and B. gestroi (a, d, e, f, h, i, I) stained with 2%
iron-propionic haematoxylin. a Spermatogonial prometaphase b Synizesis ¢ Pachytene, X and Y = sex
chromosomes d Diffuse stage e-f Early diakinesis g-h Diakinesis i Metaphase I j Anaphase I k Meta-
phase II, Y sex chromosome is negatively heteropycnotic | Anaphase II. Arrows indicate sex chromosomes.

pI] = pseudo-trivalent. N = nucleolus. Bar = 10 pm.

The significance of cytogenetics for the study of karyotype evolution and taxonomy... 119

valents divide reductionally, whereas the sex chromosomes do so equationally (Fig.
2j). All telophase I nuclei exhibit 16 chromosomes in each pole (13 + X,X,Y). The
second meiotic division follows without an interkinesis stage. At metaphase II, the
13 autosomes dispose forming a ring and in the centre of it, the sex chromosomes
are associated in a pseudo-trivalent. The Y chromosome is negatively heteropycnotic
and is oriented towards the opposite spindle pole to that of X, and X, (Fig. 2k). At
anaphase II, 14 chromosomes migrate to one pole (13 + Y) and 15 to the opposite
one (13 + X,X,) (Fig. 21).

Chromosome bandings

C-banding reveals differences in the amount and location of heterochromatin among
the three species analysed. In B. elongatum, very large C-positive blocks can be de-
tected terminally on all bivalents from prophase I to metaphase I, and interstitial dots
are also observed (Fig. 3a—c). In B. gestroi, in contrast, C-positive bands are very small
and are always located terminally (Fig. 3d, e). The results observed in B. dentatum
matched data previously described by Papeschi (1991) with C-positive bands termi-
nally located in all bivalents (Fig. 3f, g). Furthermore, the two X chromosomes in the
three species show terminally located bands, whereas the Y chromosome is C-negative
(Fig. 3a, cg).

All chromosomes stain homogenously with both fluorochromes on mitotic and
meiotic metaphase cells in the three species, except for one of the medium-sized auto-
somal bivalents in B. dentatum (Fig. 4a—c) and B. elongatum (Fig. 4d—f), and one of the
large-sized in B. gestroi (Fig. 4g—i), which show a DAPI negative/ CMA, positive band

at one terminal position.

Location of rDNA

In chromosome preparations of B. dentatum, FISH experiments with the 18S rDNA
probe show a cluster of rDNA genes located at one end of two homologous chromo-
somes each (Fig. 5a). A single cluster of signals is observed in an autosomal bivalent
at pachytene (Fig. 5b). During diffuse stage, hybridization signals are observed in the
decondensed mass of autosomal chromatin, whereas the sex chromosomes remain con-
densed forming a conspicuous DAPI bright chromatin body without any signals (Fig.
5c). At diakinesis-metaphase I, one medium-sized autosomal bivalent show hybridiza-
tion signals at both ends (Fig. 5d). In concordance with the results of B. dentatum, in
mitotic metaphases of B. elongatum and B. gestroi, hybridization signals are detected in
two homologous autosomes (Fig. 5e, g). At diakinesis-metaphase I, a single cluster of
rRNA genes is located at both ends of a medium-sized autosomal bivalent of B. elon-

gatum (Fig. 5e-f) and of a one large-sized of B. gestroi (Fig. 5h).

120 Chirino Ménica Gabriela et al. / Comparative Cytogenetics 7(2): 11 1-129 (2013)

Figure 3. C-banding in chromosomes of B. elongatum (a=-c), B. gestroi (d, e) and B. dentatum

(f, g) stained with DAPI. a Diakinesis, conspicuous terminal C-positive blocks are observed in all
autosomal bivalents and both X chromosomes b A detail of autosomal bivalents with interstitial C-
positive dots (arrows) at early diakinesis ¢ Late diakinesis d Diakinesis, small terminal C-positive
bands in some autosomal bivalents (arrows) € Metaphase I f Late diakinesis, terminal C-positive
bands in all autosomal bivalents and both X chromosomes g Metaphase II. a, c=g The Y chromosome

is C-negative. X, Y = sex chromosomes. Bar = 10 um.

The significance of cytogenetics for the study of karyotype evolution and taxonomy...

Figure 4. DAPI (blue) and CMA, (green) fluorescent banding in chromosomes of B. dentatum (ac),
B. elongatum (d-f) and B. gestroi (g-i). a Oogonial metaphase (2n = 30 = 26 + X,X,X,X,) b Diakinesis €
Metaphase II d Spermatogonial metaphase (2n = 29 = 26 + X,X,Y) e Diakinesis f Metaphase II g Sper-
matogonial metaphase (2n = 29 = 26 + X,X,Y) h Diakinesis i Metaphase I. Arrows indicate DAPI nega-

tive/ CMA, positive bands. Arrowheads show sex chromosomes (d, g). X, Y = sex chromosomes. plII =

pseudo-trivalent. Bar = 10 ym.

122 Chirino Ménica Gabriela et al. / Comparative Cytogenetics 7(2): 111-129 (2013)

Figure 5. Location of rDNA genes in chromosomes of B. dentatum (ad), B. elongatum (e, f) and B.
gestroi (g, h) by FISH with 18S rDNA probes (red signals, arrows). Chromosomes were counterstained
with DAPI (blue). a Spermatogonial anaphase (2n = 29 = 26 + X,X,Y) b Pachytene € Diffuse stage d Di-
akinesis e Spermatogonial metaphase and diakinesis f Metaphase I g Spermatogonial metaphase (2n = 29

= 26 + X,X,Y) h Diakinesis-Metaphase I. Arrowheads show sex chromosomes. Bar = 10 um.

Discussion

The Belostoma species analyzed here shared apparently similar karyotypes, since they
possess the same chromosome complement (2n = 29 = 26 + X,X,Y, male), with chro-
mosomes progressively decreasing in size. In Belostomatidae, this 2n is the modal dip-
loid chromosome number and is present in 10 species of Belostoma (Papeschi and
Bressa 2006, Chirino and Bressa 2011). On the other hand, in B. elongatum and B.
gestroi the male meiotic behaviour followed a similar pattern as previously described
for other species of this genus (Papeschi and Bidau 1985, Papeschi 1996, Bardella et al.
2012). Both B. elongatum and B. gestroi, as well as B. dentatum, showed the following
cytogenetic characteristics: a) synizesis observed, b) a multiple sex chromosome system
(XX, Y, male), c) Y chromosome negatively heteropycnotic at metaphase II, d) varia-
tion in the TCL and in the C-banding pattern, and e) a single pair of NOR-autosomes.
Within Heteroptera, the synizesis stage was described in a few species of Belostoma (Pa-
peschi and Bidau 1985, Papeschi 1992) and Dysdercus Guérin-Méneville, 1831 (Bressa
2003, Bressa et al. 2003). In this stage the chromatin condenses eccentrically in the
nucleus and chromosome pairing begins.

Heterochromatin characterization in the three species revealed differences in the
amount, distribution and location of the constitutive heterochromatin in autosomes
and both X chromosomes: i) terminal C-positive bands in B. dentatum, ii) conspicu-
ous C-positive bands at terminal and interstitial positions in B. elongatum, and iii) very
scarce C-positive bands terminally located in B. gestroi. This variation in the constitu-
tive heterochromatin of these three species could imply changes in the DNA content

The significance of cytogenetics for the study of karyotype evolution and taxonomy... 123

in the karyotype evolution in the genus, which could modify the size of the chromo-
some complement. In accordance with this suggestion, the analysis of TCL showed a
significant variation among the three species, which means that certain chromosomal
changes, must have taken place during their evolution.

Papeschi (1992) found a great interspecific variation in DNA content as well as
differences in C-positive heterochromatin percentage among B. dentatum (1.93 pg,
58.54 %), B. elongatum (1.75 pg, 59.47 %) and B. gestroi (1.13 pg, 37.24 %). Taking
into account the data previously described by Papeschi (1992), together with the re-
sults obtained from the analysis of the TCL in these three species, we propose the exist-
ence of positive relationships between TCL and DNA content and between TCL and
C-positive heterochromatin percentage (Fig. 6). Thus, it is apparent that differences in
the TCL in the three species might represent changes or variations in DNA content
since the accumulation/addition of satellite DNA in all chromosomes of the comple-
ment. The comparison between the DNA content and the C-positive heterochromatin
percentage of the three species shows a general trend, i.e. an increase in the DNA
content is accompanied by an increase in the amount of C-positive heterochromatin.
However, B. dentatum and B. elongatum have a very different DNA content and a
similar percentage of C-positive heterochromatin. On the other hand, B. gestroi shows
the lowest DNA content and the lowest C-positive heterochromatin percentage. In ac-
cordance with the earlier reports on six other species of Belostoma (Papeschi and Bidau
1985, Papeschi 1988, 1991, 1992), the genome size differences between B. dentatum
and B. elongatum could be due to a proportionate variation of both C-positive hetero-
chromatin and C-negative chromatin occurred during evolution. The chromosomes of
B. gestroi could have gained low amount of C-positive heterochromatin, or else during
evolution some C-positive bands became lost.

In Heteroptera, the classical distribution pattern of C-positive heterochromatin is
terminal in some or all chromosomes. Interstitial C-positive bands are described in a
few species and some of them correspond to secondary constrictions and NORs. In
concordance with these cytogenetic features, the C-banding pattern observed in B.
elongatum with respect to both terminal and interstitial C-positive regions agrees with
most previous reports within Belostoma (Papeschi 1995) and Heteroptera (Camacho
et al. 1985, Panzera et al. 1995, Grozeva and Nokkala 2001, Angus et al. 2004, Ituarte
and Papeschi 2004, Bressa et al. 2005, 2008, Franco et al. 2006).

The results with fluorescent banding indicate that all C-positive bands in the spe-
cies analysed were not enriched with AT or CG base pairs, as all chromosomes were
stained homogeneously with both DAPI and CMA, fluorochromes, except for the
C- positive band observed in the medium-sized autosomal bivalent of B. dentatum
and B. elongatum and in one of the large-sized of B. gestroi, which was DAPI negative/
CMA, positive. Therefore, the CMA, bright band is enriched in GC base pairs and
could represent an NOR (see below). The presence of a CMA, bright band was also
reported not only in other species of Belostoma (Papeschi and Bressa 2006) but also in
other heteropteran species, at interstitial or terminal position, either on autosomes or
sex chromosomes, and they are generally associated to NORs (Gonzalez-Garcia et al.

124 Chirino Ménica Gabriela et al. / Comparative Cytogenetics 7(2): 111-129 (2013)

C- heterochromatin (%)
DNA content (pg)
on

33 34 35 36 37 «+38 #«#939+~ «40
TCL (um) b TCL (um)

Figure 6. a Comparison between the total chromosome length (TCL) and the percentage of C-positive
heterochromatin at diakinesis in B. dentatum (58.54 + 1.27 %; circle), B. elongatum (59.47 + 0.78 %;
rectangle) and B. gestroi (37.24 + 1.50 %; diamond) b Comparison between the total chromosome length
(TCL) and the haploid DNA content (pg) in B. dentatum (1.93 + 0.16 um; circle), B. elongatum (1.75 +
0.05 um; rectangle) and B. gestroi (1.13 + 0.13 um; diamond). Data of percentage of C-positive hetero-
chromatin and the haploid DNA content were obtained from Papeschi (1991, 1992).

1996, Papeschi et al. 2001, 2003, Rebagliati et al. 2003, Cattani et al. 2004, Grozeva
et al. 2004, Poggio et al. 2011).

In Belostomatidae, the location of NORs was previously analysed by FISH with
18S rDNA probe in B. oxyurum (Dufour, 1863) (2n = 6 + XY, NOR in sex chro-
mosomes), B. micantulum (Stal, 1860) (2n = 14 + XY, NOR in sex chromosomes),
B. elegans (Mayr, 1871) (2n = 26 + X,X,Y, NOR in a pair of autosomes) (Papeschi
and Bressa 2006), and Lethocerus patruelis (Stal, 1854) (2n = 22 + 2m + XY, NOR in
sex chromosomes) (Kuznetsova et al. 2012). The present paper presents information
about the number and chromosomal location of ribosomal clusters in B. dentatum, B.
elongatum and B. gestroi, which have a single cluster located in an autosomal pair. In
these three species the NOR is associated with a CMA,-bright band. ‘The results of the
fluorescent banding and FISH in these species agree with those described for B. oxyu-
rum, B. micantulum and B. elegans, in which the NOR regions are colocalized with a
CMA. -positive band and, therefore, the repeating unit of rDNA is GC-rich (Papeschi
and Bressa 2006). Taking into account the data on the number and location of rDNA
clusters along with the type of sex chromosome systems in Belostomatidae, we can
observe two different patterns of rDNA distribution. The NOR is located at terminal
position on both sex chromosomes in species that have a simple sex chromosome sys-
tem (XY), or, in contrast, the NOR is placed at terminal position on an autosomal pair
in the species with a multiple sex chromosome system (X,X,Y). Hence, our present
results led us to propose that in Belostomatidae the location of rDNA genes could be
associated with variants of the sex chromosome systems. Moreover, this relationship
between the number and location of the NOR and the sex chromosome system has
only been observed in this family of Heteroptera.

Previous cytogenetic data on worldwide Belostomatidae species allowed Papeschi
and Bressa (2006) to propose an ancestral male karyotype 2n = 28 = 26 + XY, from

The significance of cytogenetics for the study of karyotype evolution and taxonomy... 125

which the karyotypes with multiple sex chromosome systems (2n = 26 + X,X,Y) and
those with a low 2n (6 + XY, 14 + XY, 13 + XY, 22 + XY) would have originated by
fragmentation of the ancestral X chromosome and chromosomal fusions, respectively. It
is generally accepted that multiple sex chromosome systems in Heteroptera are the result
of fragmentation(s) of the X and/or Y chromosome(s) of an ancestral simple sex chromo-
some system (Heizer 1950, Hughes-Schrader and Schrader 1961, Ueshima 1979, Man-
na 1984, Papeschi 1996, Papeschi and Bressa 2006). The holokinetic nature of heterop-
teran chromosomes and the achiasmatic male meiosis of sex chromosomes are the main
facts that support this hypothesis and may account for the variability (Ueshima 1979,
Manna 1984, Thomas 1987). In most cases of multiple sex chromosomes, the increase in
the number of sex chromosomes is not accompanied by a reduction in the number of au-
tosomes. The analysis of different populations of B. orbiculatum Estévez and Polhemus,
2001 (Papeschi 1996), B. plebejum (Stal, 1858) (Papeschi 1994), B. dilatatum (Dufour,
1863) (Bardella et. al 2012), Oechalia pacifica (Stal, 1859) (Heizer 1950) and Banasa
zeteki Sailer, 1959 (Pentatomidae) (Schrader and Hughes-Schrader 1958), polymorphic
for the sex chromosome systems, lend support to the hypothesis of a fragmentation
origin of a multiple sex chromosome systems. In all these examples one chromosome of
the simple system (XY) was replaced by two chromosomes of smaller size in the mutant
individuals. Therefore, these interspecific polymorphisms of sex chromosomes represent
a direct evidence of the origin of multiple sex chromosome system through fragmenta-
tion in Belostoma. On the other hand, the species of this genus with reduced chromo-
some numbers are characterized by a larger chromosome size, a low DNA content and
very scarce C-positive heterochromatin (Papeschi and Bressa 2006). These karyotypes
probably originated from the ancestral chromosome complement through chromosome
fusions. The possibility of their occurrence is supported by the fact that the autosomal
fusions have been found in heterozygous condition in natural populations of B. plebejum
(Papeschi 1994), Triatoma infestans (Klug, 1834) (Poggio et al. 2013) and Mepraia ga-
jardoi Frias, Henry and Gonzalez, 1998 (Pérez et al. 2004) (Reduviidae).

Published data on karyotype evolution in species of this genus (Papeschi and Bressa
2006) along with the present results of rD NA-FISH support the hypothesis that in the
ancestral male karyotype (2n = 28 = 26 + XY) the NOR would have been located in a
pair of autosomes (Fig. 7). A fragmentation of the single X chromosome in the ances-
tral karyotype resulted in multiple X chromosomes and led to a karyotype with 2n =
2926 EXER NS but keeping the ancestral NOR-autosome pair, as represented by B.
dentatum, B. elegans, B. elongatum and B. gestroi (Fig. 7). On the other hand, autosomal
fusions and the fusion of the ancestral sex chromosome pair (XY) with the autosomes
carrying the NOR would result in the reduction in the diploid chromosome (2n = 8,
16), increasing the chromosomes size and led to the presence of rDNA genes in both
X and Y chromosomes. Alternatively, the rDNA gene cluster could have been translo-
cated from an autosomal location to the X and Y chromosomes, as it has been proposed
in Dysdercus albofasciatus Berg, 1878 (Bressa et al. 2009). Furthermore, future studies
within Belostoma about this regular pattern will shed light on the karyotype evolution
within the genus and also support the mechanisms involved in the karyotype evolution.

126 Chirino Ménica Gabriela et al. / Comparative Cytogenetics 7(2): 111-129 (2013)

Ancestral karyotype
2n = 26 + XY

NOR-autosome pair

X chromosome

fragmentation autosomal and/or

sex chromosomes-autosomal
fusions (several times)

2n=6+ XY
2n = 26 + X.X,Y NOR-sex chromosome pair
NOR-autosome pair 2n= 14+ XY (B. oxyurum)
(B. dentatum, B. elegans, B. elongatum, B. gestroi) NOR- sex chromosome pair autosomal
fusion
(B. micantulum)
X chromosome sean
f tati chromosome
ey eee fragmentation 2n=13 + XY
2n = 26 + X,X,X,Y (Bplebejum
mes 2n=14+XXY
(B. dilatatum)

(B. orbiculatum, B. plebejurn)

Figure 7. Hypothetical scheme of karyotype evolution in species of the genus Belostoma. See text and
Table 1 for details.

Conventional taxonomy of water bugs has focused almost entirely on adult
specimens. There are relatively few publications on interspecific differences among
the larvae, and fewer still concern South American species. The literature of the
genus Belostoma includes much confusion because, in many cases, the species are
very similar in coloration and appearance and only males or rarely only females can
be identified (Heckman 2011). In B. dentatum, B. elongatum and B. gestroi, the
karyotype analyses allow us to get a detailed characterization and a better knowledge
of their chromosomal structure. Hence, we conclude that the cytogenetic studies
provide valuable features that can be used to solve problems on taxonomic identifi-
cation, at least for this genus.

Acknowledgements

This work was funded by grants UBACyT W917 of University of Buenos Aires, PIP
0281 of Consejo Nacional de Investigaciones Cientificas y Técnicas (CONICET), and
PICT 2007-00635 of ANPCyT from Argentina. MG Chirino and MJ Bressa thank
CONICET and ANPCyT. We wish to thank A. Bachmann and C. Armtia de Reyes

for taxonomic identification of the specimens included in the study.

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